A basic principle of flow cytometry is the passage of particles in a fluid-stream through a focused beam of laser-radiation. The particles, particularly biological cells, can be detected, identified, counted, and sorted. Cell components are fluorescently labelled and then illuminated by the laser-radiation. Scattered and emitted radiation can be measured to determine the quantity and types of cells present in a sample.
Several detectors are carefully placed around the point where the fluid-stream passes through the focused laser-beam. The suspended particles, which may range in size from 0.2 micrometers (μm) to 150 μm, pass through the focused laser-beam and scatter the laser-radiation. The fluorescently-labelled cell components are also excited by the focused laser-beam and emit radiation (fluorescence) at a longer wavelength than that of the laser-beam. This combination of scattered and fluorescent radiation is measured by the detectors. Measurement data is then analyzed, using special software, by a computer that is attached to the flow cytometer. Thousands of particles per second can be measured and analyzed.
It is generally accepted that the above described flow cytometry process is more flexible and more accurate when more wavelengths of laser-radiation are included in the laser-beam. In practice, this is accomplished by combining component laser-beams from different lasers along a common path to provide a combined laser-beam that is focused into the fluid-stream. Diode-laser modules are typically used for providing the component laser-beams. Commercially available diode-laser modules can provide laser-radiation at fundamental wavelengths ranging from the near ultraviolet to the near infrared.
An increasing number and range of wavelengths presents significant problems in the design and construction of an optical objective for focusing the combined laser-beam into the fluid-stream. It is generally accepted that for focusing two significantly different wavelengths (for example, red and blue) at a common location (focal plane) a focusing objective must include at least two optical-elements having different spectral dispersion. For example, two lenses made of different materials having high and low spectral dispersion. An objective arranged to focus two different wavelengths in a common focal plane is generally referred to as an “achromatic objective”.
If three significantly different wavelengths (for example, red, green, and blue) are to be focused at a common location, a focusing objective must include at least three optical-elements having different spectral dispersion. An objective arranged to focus three significantly different wavelengths in a common focal plane is generally referred to as an “apochromatic objective”.
In achromatic or apochromatic objectives, individual optical-elements (singlets) of different spectral dispersion may need to be “cemented” together in forms referred to by practitioners of the lens design art as “doublets” or “triplets”. These forms can be problematic when ultraviolet wavelengths are included in a flow cytometer, as optical cements (adhesives) may be degraded by ultraviolet laser-radiation.
Based on conventional optical design wisdom, it is expected that the objective required to focus the laser-beam into the fluid-stream will become even more complex and more expensive as additional wavelengths of laser-radiation are included in a flow cytometer. For example, a flow cytometer having four or more wavelengths. This could result in the complexity and cost of a focusing objective imposing a practical upper limit to the number of wavelengths of laser-radiation that can be used in a flow cytometer.
There is need for a simple focusing objective, capable of focusing four or more laser-radiation wavelengths in a common focal plane, but wherein the number of different optical materials (glasses) required is less than the number of different wavelengths to be focused in the common focal plane. Preferably, the focusing objective should not include any cemented doublet or triplet elements. Preferably, the focusing objective will create a combined laser-beam in the common focal plane that is insensitive to any fluctuations of beam-parameters of the component laser-beams from different lasers.